IBD-associated microbial nucleic acid molecules

ABSTRACT

The present invention provides nucleic acid and amino acid sequence of the novel I-1 and I-2 polypeptides, which are associated with human inflammatory bowel disease (IBD). Methods of diagnosing and treating inflammatory bowel disease using the IBD-associated I-1 and I-2 antigens also are provided.

[0001] This application is a continuation of application Ser. No.09/820,576, filed Mar. 28, 2001, which is a divisional of applicationSer. No. 09/303,120, filed Apr. 30, 1999.

ACKNOWLEDGMENT

[0002] This work was supported by grant number DK46763 awarded by theNational Institutes of Health. The United States government has certainrights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention relates generally to the fields of immunology,microbiology and inflammatory bowel disease and more specifically to thediagnosis and treatment of inflammatory bowel disease using microbialantigens.

[0005] 2. Background Information

[0006] Inflammatory bowel disease (IBD) is the collective term used todescribe two gastrointestinal disorders of unknown etiology: Crohn'sdisease (CD) and ulcerative colitis (UC). The course and prognosis ofIBD, which occurs world-wide and is reported to afflict as many as twomillion people, varies widely. Onset of IBD is predominantly in youngadulthood with diarrhea, abdominal pain, and fever the three most commonpresenting symptoms. The diarrhea may range from mild to severe, andanemia and weight loss are additional common signs of IBD. Ten percentto fifteen percent of all patients with IBD will require surgery over aten year period. In addition, patients with IBD are at increased riskfor the development of intestinal cancer. Reports of an increasedoccurrence of psychological problems, including anxiety and depression,are perhaps not surprising symptoms of what is often a debilitatingdisease that strikes people in the prime of life.

[0007] Unfortunately, the available therapies for inflammatory boweldisease are few, and both diagnosis and treatment have been hampered bya lack of knowledge regarding the etiology of the disease. What isclear, however, is that a combination of genetic factors, exogenoustriggers and endogenous microflora can contribute to the immune-mediateddamage to the intestinal mucosa seen in inflammatory bowel disease. InCrohn's disease, bacteria have been implicated in initiation andprogression of the disease: the intestinal inflammation in Crohn'sdisease is notable for its frequent responsiveness to antibiotics andsusceptibility to bacterial fecal flow. Common intestinal colonists andnovel pathogens have been implicated in Crohn's by direct detection orby disease associated anti-microbial immune responses. Furthermore, inmany genetically susceptible animal models of chronic colitis, lumenalmicro-organisms are a necessary cofactor for disease; animals housed ina germ-free environment do not develop colitis. However, despite muchdirect and indirect evidence for a role for enteric microorganisms inCrohn's disease, the pathogenic organisms and antigens contributing tothe immune dysregulation seen in this disease have not been identified.

[0008] Current diagnostic assays for Crohn's disease are unable todetect all patients with the disease. Thus, identification of novelmicrobial antigens associated with Crohn's disease would providereagents that can increase the sensitivity of current diagnostic assays.In addition, such microbial antigens can bear a disease related T-cellepitope and, as original or contributing inducers of the disease-relatedimmune response, can be effective tolerogenic antigens for treatinginflammatory bowel disease. Identification of IBD-associated microbialantigens also would facilitate isolation of the involved microbialspecies, paving the way for the discovery of new antibiotics or drugsfor treating inflammatory bowel disease, such drugs ameliorating diseaseby eliminating the microbial inducers of disease.

[0009] Thus, there is a need for identification and isolation ofmicrobial IBD-associated antigens for diagnosing and treating the manyindividuals suffering from inflammatory bowel disease. The presentinvention satisfies this need by providing the IBD-associated I-1 andI-2 microbial antigens. Related advantages are provided as well.

SUMMARY OF THE INVENTION

[0010] The present invention provides an isolated inflammatory boweldisease-associated I-2 polypeptide having substantially the same aminoacid sequence as SEQ ID NO: 2. The invention also provides an isolatedimmunoreactive fragment of an I-2 polypeptide having substantially thesame amino acid sequence as a portion of SEQ ID NO: 2. An isolatedimmunoreactive fragment of an I-2 polypeptide can have, for example, atleast ten contiguous amino acids of SEQ ID NO: 2.

[0011] Also provided by the present invention is substantially purifiedantibody material that selectively binds an I-2 polypeptide having SEQID NO: 2. Such a substantially purified antibody material can be, forexample, substantially purified polyclonal or monoclonal antibodymaterial.

[0012] The invention further provides an isolated nucleic acid moleculehaving a nucleic acid sequence encoding substantially the same aminoacid sequence as SEQ ID NO: 2. An isolated nucleic acid molecule of theinvention can have, for example, the nucleic acid sequence SEQ ID NO: 1.

[0013] Also provided by the invention is a method of diagnosinginflammatory bowel disease (IBD) in a subject. The method includes thesteps of obtaining a sample from the subject; contacting the sample withan I-2 polypeptide, or immunoreactive fragment thereof, under conditionssuitable to form a complex of the I-2 polypeptide, or the immunoreactivefragment thereof, and antibody to the I-2 polypeptide; and detecting thepresence or absence of the complex, where the presence of the complexindicates that the subject has IBD. A method of the invention fordiagnosing inflammatory bowel disease can be useful, for example, fordiagnosing Crohn's disease. In a method of the invention for diagnosinginflammatory bowel disease, the presence or absence of the complex canbe detected, for example, with a detectable secondary antibody that hasspecificity for a class determining portion of the antibody to the I-2polypeptide.

[0014] Further provided by the invention is a method of inducingtolerance in a patient with inflammatory bowel disease by administeringan effective dose of an I-2 polypeptide, or tolerogenic fragmentthereof, to the patient with IBD. The methods of the invention can beparticularly useful for treating a patient having Crohn's disease. In amethod of the invention for inducing tolerance, the I-2 polypeptide tobe administered can have, for example, the amino acid sequence of SEQ IDNO: 2.

[0015] The invention also provides a composition including an I-2polypeptide having substantially the same amino acid sequence as SEQ IDNO: 2, or tolerogenic fragment thereof, combined with a tolerogizingmolecule. In a composition of the invention, the I-2 polypeptide canhave, for example, the amino acid sequence SEQ ID NO: 2. A tolerogenicfragment useful in a composition of the invention can have, for example,at least ten contiguous amino acids of SEQ ID NO: 2.

[0016] The present invention also provides a method of identifying anagent useful in treating inflammatory bowel disease. The method includesthe steps of obtaining a specimen of an enteric microbe from a patientwith inflammatory bowel disease; isolating from the specimen a microbialspecies that includes a nucleic acid molecule encoding an I-2polypeptide; contacting the microbial species with an agent; andassaying for reduced growth or viability of the microbial species ascompared to the growth or viability in the absence of the agent, wherethe reduced growth or viability of the microbial species indicates thatthe agent is an agent useful in treating inflammatory bowel disease. Amethod of the invention can be useful, for example, for identifying anagent for treating Crohn's disease. The methods of the invention can beparticularly useful for screening agents which are antibiotics.

[0017] The invention additionally provides a method of identifying anagent useful in treating inflammatory bowel disease using a novel animalmodel. The method includes the steps of administering an I-2 polypeptideto a non-human animal, whereby one or more symptoms of IBD areexhibited; administering an agent to the non-human animal; and assayingthe level of the one or more symptoms characteristic of IBD, where areduction in the level of the one or more symptoms as compared to acontrol level indicates that the agent is an agent useful in treatingIBD. The methods of the invention can be applied, for example, toidentification of agents useful in treating Crohn's disease. The I-2polypeptide administered can have, for example, the amino acid sequenceSEQ ID NO: 2. A non-human animal particularly useful in the methods ofthe invention can be, for example, a mouse deficient in Gai2, TCRA orIL-10.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows the I-1 and I-2 nucleic acid sequences and encodedpolypeptides. A. Shown are the I-2 nucleic acid sequence (SEQ ID NO: 1)and predicted amino acid sequence (SEQ ID NO: 2). B. Shown are the I-1nucleic acid sequence (SEQ ID NO: 3) and I-1 open reading frame 1 (top;SEQ ID NO: 4) and open reading frame 2 (bottom; SEQ ID NO: 5).

[0019]FIG. 2 shows an alignment of the I-2 amino acid sequence SEQ IDNO: 2 with homologous sequences. Sequences were identified by tBLASTxsearch against the non-redundant database. Alignments were generated byCLUSTALW, with modifications by Genedoc. “Clostridium” is the predictedprotein 4 from Clostridium pasteurianum (SEQ ID NO: 6; accession number481591); “Mycobacterium” is the predicted protein Rv3557c fromMycobacterium tuberculosis (SEQ ID NO: 7; accession number 1877288); and“Aquifex” is a transcriptional regulator from Aquifex aeolicus (SEQ IDNO: 8; accession number 2984362). Residues identical or conservativelysubstituted among four polypeptides are shown in black; residuesidentical or conservatively substituted among three polypeptides areshown in dark gray; and residues identical or conservatively substitutedbetween two polypeptides are shown in light gray. Consensus residues areshown beneath the alignment, with one-letter codes indicating conservedamino acids; “6” indicating a conserved non-polar (hydrophobic) residuesuch as leucine, isoleucine, alanine, valine or methionine; and “5”indicating the conserved aromatic residue phenylalanine or tyrosine.

[0020]FIG. 3 shows the results of PCR analysis with I-2 (SEQ ID NO: 1)specific primers using paraffin embedded colonic samples from CDpatients, UC patients and control individuals. “N” designates the numberof samples assayed, and “N+” designates the number of positive samples.“Ca” designates cancer resection samples; “Divertic” designatesdiverticulitis samples; “append” designates appendicitis samples; and“I-col “designates ischemic colitis.

[0021]FIG. 4 shows IgG and IgA reactivities to the I-2 polypeptide (SEQID NO: 2) in various populations.

[0022] A. Sera from Crohn's disease patients and normal individuals wasassayed by ELISA at the indicated dilutions for IgG reactivity to theGST-I-2 fusion polypeptide (SEQ ID NO: 2). The absorbance values werecalculated by subtracting absorbance to GST alone.

[0023] B. Sera from Crohn's disease, ulcerative colitis and normalindividuals was assayed by ELISA for IgA reactivity to the GST-I-2fusion polypeptide (SEQ ID NO: 2). The absorbance values were calculatedby subtracting absorbance to GST alone. The cut-off (dotted line) wasset as two standard deviations above the mean value for the normalpopulation.

[0024]FIG. 5 shows T cell proliferation assays using CD4+ T cellsderived from normal C57BL/6J mice. T cells were stimulated with theindicated concentration of GST-I-2 fusion protein, shown on the Y axis.The X axis represents counts of incorporated [³H]-thymidine. SEBrepresents T cells stimulated with the positive control Staphylococcusaureus enterotoxin B.

[0025]FIG. 6 shows T cell cytokine expression in response to challengewith the GST-I-2 fusion protein.

[0026]FIG. 7 shows a variety of conditions for culturing microbialorganisms isolated from IBD patients.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The pathogenesis of inflammatory bowel disease, although poorlyunderstood, ultimately involves immune-mediated tissue damage. Similarto autoimmune disorders such as diabetes mellitus and multiplesclerosis, inflammatory bowel disease is associated with variousimmunologic abnormalities and can represent a process of immunedysfunction. However, unlike the other disorders, inflammatory boweldisease occurs in a mucosal site interfacing with the intestinal lumen,and, therefore, a primary immune target in inflammatory bowel diseasecan be extrinsic agent such as a chronic microbial colonist. In thiscase, the mucosal injury characteristic of inflammatory bowel disease isa consequence of inflammatory bystander damage to resident parenchymalcells.

[0028] The present invention is directed to the exciting discovery thatseveral microbial DNA sequences are found preferentially in involvedCrohn's disease (CD) mucosa as compared to uninvolved mucosa. Asdisclosed herein, representational difference analysis (RDA), a PCRdriven subtractive cloning approach for identifying DNA sequences foundpreferentially in an infected area, was used to isolate DNA sequencesfrom a Crohn's disease patient that were differentially present inmononuclear cells from the lamina propria in an area with ulcerations ascompared to an area macroscopically free of disease (see Example I). Asdisclosed herein, two IBD-associated sequences were of microbial origin.The nucleic acid and amino acid sequences, designated I-1 (SEQ ID NOS: 3to 5) and I-2 (SEQ ID NOS: 1 and 2) are shown in FIG. 1. As furtherdisclosed herein, PCR analysis of colonic samples from CD, UC andnon-IBD patients revealed that the I-2 sequence (SEQ ID NO: 1) was moreoften found in involved CD tissue than in UC or non-IBD samples (ExampleI and FIG. 3). Thus, novel microbial sequences have been identified thatare associated with inflammatory bowel disease, in particular, withinflamed CD lesions. Isolation of microbial sequences associated withIBD implicates microbes in the pathogenesis of IBD and provides valuablereagents for diagnosing or ameliorating inflammatory bowel disease.

[0029] Thus, the present invention provides an isolated inflammatorybowel disease associated I-2 polypeptide having substantially the sameamino acid sequence as SEQ ID NO: 2. The invention also provides anisolated immunoreactive fragment of an I-2 polypeptide havingsubstantially the same amino acid sequence as a portion of SEQ ID NO: 2.An isolated immunoreactive fragment of an I-2 polypeptide can have, forexample, at least ten contiguous amino acids of SEQ ID NO: 2.

[0030] The term “isolated,” as used herein in reference to a polypeptidemeans a polypeptide that is in a form that is relatively free fromcontaminating lipids, polypeptides, nucleic acids or other cellularmaterial normally associated with the polypeptide in a cell.

[0031] As used herein, the term “I-2 polypeptide” means a polypeptidehaving substantially the same amino acid sequence as the microbial I-2polypeptide (SEQ ID NO: 2) shown in FIG. 1A. The microbial I-2polypeptide (SEQ ID NO: 2) is a polypeptide of 100 amino acids sharingsome similarity to bacterial transcriptional regulators, with thegreatest similarity in the amino-terminal 30 amino acids. As illustratedin FIG. 2, the I-2 polypeptide shares weak homology with the predictedprotein 4 from C. pasteurianum (SEQ ID NO: 6); Rv3557c fromMycobacterium tuberculosis (SEQ ID NO: 7); and a transcriptionalregulator from Aquifex aeolicus (SEQ ID NO: 8). As disclosed in ExampleI, the I-2 encoding nucleic acid (SEQ ID NO: 1) is differentiallypresent in involved Crohn's disease tissue, as compared to mucosamacroscopically free of disease.

[0032] An I-2 polypeptide having substantially the same amino acidsequence as SEQ ID NO: 2 can be the naturally occurring I-2 polypeptide(SEQ ID NO: 2) or a related polypeptide having substantial amino acidsequence similarity to this sequence. Such related polypeptides exhibitgreater sequence similarity to the I-2 polypeptide SEQ ID NO: 2 than tothe C. pasteurianum sequence SEQ ID NO: 6 and include isotype variantsor homologs of the amino acid sequence shown in FIG. 1A. As used herein,the term I-2 polypeptide generally describes polypeptides generallyhaving an amino acid sequence with greater than about 50% identity,preferably greater than about 60% identity, more preferably greater thanabout 70% identity, and can be a polypeptide having greater than about80%, 90%, 95%, 97%, or 99% amino acid sequence identity with SEQ ID NO:2, said amino acid identity determined with CLUSTALW using the BLOSUM 62matrix with default parameters. The C. pasteurianum protein 4 (SEQ IDNO: 6) has about 21% amino acid identity with the I-2 polypeptide SEQ IDNO: 2 and, therefore, is not an I-2 polypeptide as defined herein.

[0033] The I-1 nucleic acid sequence (SEQ ID NO: 3) contains two openreading frames (ORFs) on opposite strands. The top strand I-1 ORF shownin FIG. 1B encodes a predicted polypeptide of 115 amino acids (SEQ IDNO: 4) with homology to the prokaryotic transcription factor ptxR. Thebottom strand ORF encodes a predicted polypeptide of 114 amino acids(SEQ ID NO: 5). The I-1 encoding nucleic acid (SEQ ID NO: 3) also wasisolated by RDA analysis as a sequence preferentially found in involvedCD mucosa (see Example I).

[0034] An I-1 polypeptide can have substantially the same amino acidsequence as SEQ ID NO: 4 or SEQ ID NO: 5. Such an I-1 polypeptide can bea naturally occurring I-1 polypeptide (SEQ ID NOS: 4 or 5) or a relatedpolypeptide, for example, an isotype variant or homologous sequence froma different bacterial species having substantial amino acid sequencesimilarity to one of these sequences. As used herein, the term I-1polypeptide generally describes polypeptides generally having an aminoacid sequence with greater than about 50% identity, preferably greaterthan about 60% identity, more preferably greater than about 70%identity, and can be a polypeptide having greater than about 80%, 90%,95%, 97%, or 99% amino acid sequence identity with SEQ ID NOS: 4 or 5,said amino acid identity determined with CLUSTALW using the BLOSUM 62matrix with default parameters.

[0035] As used herein, the term “substantially the same amino acidsequence,” when used in reference to an I-1 or I-2 polypeptide, isintended to mean a sequence as shown in FIG. 1A or FIG. 1B, or asimilar, non-identical sequence that is considered by those skilled inthe art to be a functionally equivalent amino acid sequence. Forexample, an amino acid sequence that has substantially the same aminoacid sequence as an I-1 polypeptide (SEQ ID NOS: 4 or 5) or I-2polypeptide (SEQ ID NO: 2) can have one or more modifications such asamino acid additions, deletions or substitutions relative to the aminoacid sequence of SEQ ID NO: 4 or 5, or SEQ ID NO: 2, respectively,provided that the modified polypeptide retains substantially at leastone biological activity of I-1 or I-2 such as immunoreactivity ortolerogenic activity, described further below. Comparison forsubstantial similarity between amino acid sequences is usually performedwith sequences between about 6 and 100 residues, preferably betweenabout 10 and 100 residues and more preferably between about 25 and 35residues.

[0036] Thus, it is understood that limited modifications can be made toan I-1 or I-2 polypeptide, or to an immunoreactive or tolerogenicfragment thereof, as described further below, without destroying itsbiological function. A modification of an I-1 or I-2 polypeptide thatdoes not destroy immunoreactivity or a modification of an I-1 or I-2polypeptide that does not destroy tolerogenic activity is encompassedwithin the meaning of the term I-1 polypeptide, or I-2 polypeptide, asused herein. A modification can be, for example, an addition, deletion,or substitution of one or more amino acid residues; substitution of acompound that mimics amino acid structure or function; or addition ofchemical moieties such as amino or acetyl groups. The activity of amodified I-1 or I-2 polypeptide or fragment thereof can be assayed, forexample, using one of the assays for immunoreactivity or tolerogenicactivity disclosed herein (see below).

[0037] A particularly useful modification of a polypeptide of theinvention, or fragment thereof, is a modification that confers, forexample, increased stability. Incorporation of one or more D-amino acidsis a modification useful in increasing stability of a polypeptide orpolypeptide fragment. Similarly, deletion or substitution of lysine canincrease stability by protecting against degradation. For example, sucha substitution can increase stability and, thus, bioavailability of oneof the polypeptide antigens disclosed herein.

[0038] The I-2 polypeptide, and fragments thereof, can be useful toprepare substantially purified antibody material that selectively bindsan I-2 polypeptide (SEQ ID NO: 2). The antibody material can be, forexample, substantially purified polyclonal antiserum or monoclonalantibody material. The antibody material of the invention be useful, forexample, in determining the presence and location of I-2 polypeptidewithin the mucosa of afflicted patients and in diagnosing inflammatorybowel disease. The substantially purified antibody material of theinvention can be useful, for example, in an ELISA orimmunohistopathological assay for diagnosing Crohn's disease.

[0039] As used herein, the term “antibody material” is used in itsbroadest sense to include polyclonal and monoclonal antibodies, as wellas polypeptide fragments of antibodies that retain a selective bindingactivity for an I-2 polypeptide of at least about 1×10⁵ M⁻¹. One skilledin the art would know that anti-I-2 antibody fragments such as Fab,F(ab′)₂ and Fv fragments can retain selective binding activity for anI-2 polypeptide and, thus, are included within the definition of anantibody. In addition, the term antibody material as used hereinencompasses non-naturally occurring antibodies and fragments containing,at a minimum, one VH and one VL domain, such as chimeric antibodies,humanized antibodies, single chain Fv fragments (scfv) that selectivelybind an I-2 polypeptide. Such non-naturally occurring antibodies can beconstructed using solid phase peptide synthesis, produced recombinantlyor obtained, for example, by screening combinatorial librariesconsisting of variable heavy chains and variable light chains asdescribed by Borrebaeck (Ed.), Antibody Engineering (Second edition) NewYork: Oxford University Press (1995), which is incorporated herein byreference.

[0040] Antibody material “selective for” an I-2 polypeptide, or that“selectively binds” an I-2 polypeptide, binds with substantially higheraffinity to that polypeptide than to an unrelated polypeptide. Thesubstantially purified antibody material of the invention also can bespecific for an I-2 polypeptide, whereby its binding affinity issignificantly higher for an I-2 polypeptide than for relatedpolypeptides such as SEQ ID NOS: 6 to 8.

[0041] Anti-I-2 antibody material can be prepared, for example, using anI-2 fusion protein or a synthetic peptide encoding a portion of the I-2polypeptide (SEQ ID NO: 2) as an immunogen. One skilled in the art wouldknow that purified I-2 polypeptide, which can be produced recombinantly,or fragments of I-2, including peptide portions of I-2 such as syntheticpeptides, can be used as an immunogen. Non-immunogenic fragments orsynthetic peptides of I-2 can be made immunogenic by coupling the haptento a carrier molecule such as bovine serum albumin (BSA) or keyholelimpet hemocyanin (KLH). In addition, various other carrier moleculesand methods for coupling a hapten to a carrier molecule are well knownin the art as described, for example, by Harlow and Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1988), which isincorporated herein by reference.

[0042] The term “substantially purified,” as used herein in reference toantibody material, means that the antibody material is substantiallydevoid of polypeptides, nucleic acids and other cellular material whichwith an antibody is normally associated in a cell. The claimed antibodymaterial that selectively binds an I-2 polypeptide (SEQ ID NO: 2)further is substantially devoid of antibody material of unrelatedspecificities, i.e. that does not selectively bind an I-2 polypeptide.The antibody material of the invention can be prepared in substantiallypurified form, for example, by I-2 affinity purification of polyclonalanti-I-2 antisera, by screening phage displayed antibodies against theI-2 polypeptide (SEQ ID NO: 2), or as monoclonal antibodies preparedfrom hybridomas.

[0043] The invention further provides an isolated nucleic acid moleculehaving a nucleic acid sequence encoding substantially the same aminoacid sequence as SEQ ID NO: 2. An isolated nucleic acid molecule of theinvention can have, for example, the nucleic acid sequence SEQ ID NO: 1.These nucleic acid molecules are useful, for example, in producingrecombinant polypeptides and as probes for detecting I-2 mRNAexpression. Nucleotide portions of SEQ ID NO: 1 also are useful, forexample, as primers for PCR analysis (see Example I).

[0044] Isolated nucleic acid molecules of the invention include, forexample, nucleic acid molecules encoding I-2 polypeptide homologs,nucleic acid molecules that are related, but different and encode thepolypeptide of SEQ ID NO: 2 due to the degeneracy of the genetic code,and nucleic acid molecules that are related, but different and encode anI-2 polypeptide different from SEQ ID NO: 2 that exhibitsimmunoreactivity or tolerogenic activity.

[0045] The methods of the invention relate to the diagnosis andtreatment of inflammatory bowel disease, which is a designation thatencompasses the broad categories of Crohn's disease and ulcerativecolitis. Crohn's disease (regional enteritis) is a disease of chronicinflammation that can involve any part of the gastrointestinal tract.Commonly the distal portion of the small intestine (ileum) and cecum areaffected. In other cases, the disease is confined to the smallintestine, colon or anorectal region. Crohn's disease occasionallyinvolves the duodenum and stomach, and more rarely the esophagus andoral cavity.

[0046] The variable clinical manifestations of Crohn's disease are, inpart, a result of the varying anatomic localization of the disease. Themost frequent symptoms of CD are abdominal pain, diarrhea and recurrentfever. CD is commonly associated with intestinal obstruction or fistula,which is an abnormal passage between diseased loops of bowel, forexample. Crohn's disease also includes complications such asinflammation of the eye, joints and skin; liver disease; kidney stonesor amyloidosis. In addition, CD is associated with an increased risk ofintestinal cancer.

[0047] Several features are characteristic of the pathology of Crohn'sdisease. The inflammation associated with CD, known as transmuralinflammation, involves all layers of the bowel wall. Thickening andedema, for example, typically also appear throughout the bowel wall,with fibrosis also present in long-standing disease. The inflammationcharacteristic of CD also is discontinuous in that segments of inflamedtissue, known as “skip lesions,” are separated by apparently normalintestine. Furthermore, linear ulcerations, edema, and inflammation ofthe intervening tissue lead to a “cobblestone” appearance of theintestinal mucosa, which is distinctive of CD.

[0048] A hallmark of Crohn's disease is the presence of discreteaggregations of inflammatory cells, known as granulomas, which aregenerally found in the submucosa. Some Crohn's disease cases display thetypical discrete granulomas, while others show a diffuse granulomatousreaction or nonspecific transmural inflammation. As a result, thepresence of discrete granulomas is indicative of CD, although theabsence of granulomas also is consistent with the disease. Thus,transmural or discontinuous inflammation, rather than the presence ofgranulomas, is a preferred diagnostic indicator of Crohn's disease(Rubin and Farber, Pathology (Second Edition) Philadelphia: J. B.Lippincott Company (1994), which is incorporated herein by reference).

[0049] Ulcerative colitis (UC) is a disease of the large intestinecharacterized by chronic diarrhea with cramping abdominal pain, rectalbleeding, and loose discharges of blood, pus and mucus. Themanifestations of ulcerative colitis vary widely. A pattern ofexacerbations and remissions typifies the clinical course of most UCpatients (70%), although continuous symptoms without remission arepresent in some patients with UC. Local and systemic complications of UCinclude arthritis, eye inflammation such as uveitis, skin ulcers andliver disease. In addition, ulcerative colitis and especiallylong-standing, extensive disease is associated with an increased risk ofcolon carcinoma.

[0050] Several pathologic features characterize UC in distinction toother inflammatory bowel diseases. Ulcerative colitis is a diffusedisease that usually extends from the most distal part of the rectum fora variable distance proximally. The term left-sided colitis describes aninflammation that involves the distal portion of the colon, extending asfar as the splenic flexure. Sparing of the rectum or involvement of theright side (proximal portion) of the colon alone is unusual inulcerative colitis. The inflammatory process of ulcerative colitis islimited to the colon and does not involve, for example, the smallintestine, stomach or esophagus. In addition, ulcerative colitis isdistinguished by a superficial inflammation of the mucosa that generallyspares the deeper layers of the bowel wall. Crypt abscesses, in whichdegenerated intestinal crypts are filled with neutrophils, also aretypical of ulcerative colitis (Rubin and Farber, supra, 1994).

[0051] In comparison with Crohn's disease, which is a patchy diseasewith frequent sparing of the rectum, ulcerative colitis is characterizedby a continuous inflammation of the colon that usually is more severedistally than proximally. The inflammation in ulcerative colitis issuperficial in that it is usually limited to the mucosal layer and ischaracterized by an acute inflammatory infiltrate with neutrophils andcrypt abscesses. In contrast, Crohn's disease affects the entirethickness of the bowel wall with granulomas often, although not always,present. Disease that terminates at the ileocecal valve, or in the colondistal to it, is indicative of ulcerative colitis, while involvement ofthe terminal ileum, a cobblestone-like appearance, discrete ulcers orfistulas suggest Crohn's disease. Characteristics that serve todistinguish Crohn's disease from ulcerative colitis are summarized inTable 1 (Rubin and Farber, supra, 1994). TABLE 1 Characteristic Featuresof Crohn's disease and ulcerative colitis Crohn's Ulcerative FeatureDisease Colitis Macroscopic Thickened bowel wall Typical UncommonLuminal narrowing Typical Uncommon “Skip” lesions Common Absent Rightcolon predominance Typical Absent Fissures and fistulas Common AbsentCircumscribed ulcers Common Absent Confluent linear ulcers Common AbsentPseudopolyps Absent Common Microscopic Transmural inflammation TypicalUncommon Submucosal fibrosis Typical Absent Fissures Typical RareGranulomas Common Absent Crypt abscesses Uncommon Typical

[0052] Certain immune-mediated disorders, including systemic lupuserythematosis, primary biliary cirrhosis and autoimmune hepatitis, areclosely associated with distinctive patterns of autoantibody production.Disease-specific marker antibodies also have been observed in IBD. Inthe case of ulcerative colitis, anti-neutrophil cytoplasmic antibodiesthat produce a perinuclear staining pattern (pANCA), for example, uponindirect immunofluorescence microscopy of alcohol-fixed neutrophils, areelevated in 68-80% of UC patients and less frequently in other disordersof the colon. In Crohn's disease, serum reactivity to the cell wallmannan polysaccharide of Saccharomyces uvarum (brewer's yeast) is aserologic marker for a majority of individuals with Crohn's disease(Sendid et al., Clin. Diag. Lab. Immunol., 3:219-226 (1996), which isincorporated herein by reference).

[0053] As disclosed herein, ELISA analysis showed increased IgG serumreactivity to a GST-I-2 fusion polypeptide (SEQ ID NO: 2) in patientswith Crohn's disease as compared to normal individuals (see FIG. 4A). Asshown in FIGS. 4B, 9 of 10 Crohn's disease patients had IgA serumreactivity to the GST-I-2 fusion polypeptide greater than two standarddeviations above the mean value for a normal population. In contrast, nonormal serum samples contained anti-I-2 IgA reactivity above thiscutoff. The anti-I-2 serum IgA reactivity also was significantly higheron average in samples from Crohn's disease patients as compared toulcerative colitis patients. These results indicate that reactivity tothe I-2 polypeptide (SEQ ID NO: 2) can be used to differentiate Crohn'sdisease from normal individuals and those with UC.

[0054] Based on the above findings, the present invention providesmethods of diagnosing inflammatory bowel disease (IBD) in a subject. Themethods include the steps of obtaining a sample from the subject;contacting the sample with an I-2 polypeptide, or immunoreactivefragment thereof, under conditions suitable to form a complex of the I-2polypeptide, or the immunoreactive fragment thereof, and antibody to theI-2 polypeptide; and detecting the presence or absence of the complex,where the presence of the complex indicates that the subject has IBD. Amethod of the invention for diagnosing inflammatory bowel disease can beuseful, for example, for diagnosing Crohn's disease. The presence orabsence of the complex can be detected, for example, with a detectablesecondary antibody that has specificity for a class determining portionof the antibody to the I-2 polypeptide.

[0055] As used herein, the term “subject” means any animal capable ofhaving inflammatory bowel disease, including a human, non-human primate,rabbit, rat or mouse, especially a human. A subject generally has one ormore symptoms of ulcerative colitis or Crohn's disease.

[0056] A sample useful in the methods of the invention can be obtainedfrom any biological fluid having antibodies such as, for example, wholeblood, plasma, saliva, or other bodily fluid or tissue, preferablyserum. A sample to be assayed according to the methods of the inventioncan be obtained from any such subject.

[0057] As used herein, the term “complex” is synonymous with “immunecomplex” and means an aggregate of two or more molecules that resultsfrom specific binding between an antigen, such as a protein or peptide,and an antibody. In the methods of the invention, a complex is formed byspecific binding of an I-2 polypeptide to an antibody.

[0058] In the methods of the invention, a complex can be detected with adetectable secondary antibody that has specificity for a classdetermining portion of the antibody to the I-2 polypeptide. Such asecondary antibody can be, for example, an anti-IgA secondary antibody,an anti-IgG secondary antibody, or a combination of anti-IgA andanti-IgG secondary antibodies.

[0059] As used herein, the term “secondary antibody” means an antibodyor combination of antibodies, which binds an antibody that specificallybinds an I-2 polypeptide having substantially the amino acid sequenceSEQ ID NO: 2. One skilled in the art understands that, preferably, asecondary antibody does not compete with the I-2 antigen for binding tothe primary antibody. A secondary antibody can bind any epitope of theantibody that specifically binds the I-2 polypeptide. A particularlyuseful secondary antibody is an anti-IgA or anti-IgG antibody havingspecificity for the class determining portion of the primary antibody. Auseful secondary antibody is specific for the species from which thesample was obtained. For example, if human serum is the sample to beassayed, mouse anti-human IgA or IgG can be a useful secondary antibody.A combination of different antibodies, which can be useful in themethods of the invention, also is encompassed within the meaning of theterm secondary antibody, provided that at least one antibody of thecombination reacts with an antibody that specifically binds an I-2polypeptide.

[0060] As used herein, the term “class determining portion,” when usedin reference to a secondary antibody, means the heavy chainconstant-region sequence of an antibody that determines the isotype,such as IgA, IgD, IgE, IgG or IgM. Thus, a secondary antibody that hasspecificity for the class determining portion of an IgA molecule, forexample, binds IgA in preference to other antibody isotypes.

[0061] A secondary antibody useful in the invention can be obtainedcommercially or by techniques well known in the art. Such an antibodycan be a polyclonal or, preferably, monoclonal antibody. For example,IgG reactive polyclonal antibodies can be prepared using IgG or Fcfragments of IgG as an immunogen to stimulate the production ofantibodies in the antisera of an animal such as a rabbit, goat, sheep orrodent, as described in Harlow and Lane, Antibodies: A Laboratory ManualNew York: Cold Spring Harbor Laboratory (1988), which is incorporatedherein by reference. Monoclonal secondary antibodies, which are apopulation of antibody molecules that contain only one species ofidiotope capable of binding a particular antigen epitope also can beproduced by routine methods (see, for example, Harlow and Lane, supra,1988) or obtained commercially.

[0062] The term “detectable secondary antibody” means a secondaryantibody, as defined above, that can be detected or measured byanalytical methods. Thus, the term secondary antibody includes anantibody labeled directly or indirectly with a detectable marker thatcan be detected or measured and used in a convenient assay such as anenzyme-linked immunosorbent assay, radioimmunoassay, radialimmunodiffusion assay or Western blotting assay. A secondary antibodycan be labeled, for example, with an enzyme, radioisotope, fluorochromeor chemiluminescent marker. In addition, a secondary antibody can berendered detectable using a biotin-avidin linkage such that a detectablemarker is associated with the secondary antibody. Labeling of thesecondary antibody, however, should not impair binding of the secondaryantibody to the I-2 polypeptide. If desired, a multiple antibody systemcan be used as the secondary antibody as discussed above. In such asystem, at least one of the antibodies is capable of binding the primaryanti-I-2 antibody and at least one of the antibodies can be readilydetected or measured by analytical methods.

[0063] A secondary antibody can be rendered detectable by labeling withan enzyme such as horseradish peroxidase (HRP), alkaline phosphatase(AP), β-galactosidase or urease, for example. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable by measuringabsorbance at 450 nm. An alkaline phosphatase detection system can beused with the chromogenic substrate p-nitrophenyl phosphate, forexample, which yields a soluble product readily detectable by measuringabsorbance at 405 nm. Similarly, a β-galactosidase detection system canbe used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a solubleproduct detectable by measuring absorbance at 410 nm, or a ureasedetection system can be used with a substrate such as urea-bromocresolpurple (Sigma Immunochemicals, St. Louis, Mo.). A secondary antibody canbe linked to an enzyme by methods well known in the art (Harlow andLane, supra, 1988) or can be obtained from a number of commercialsources. For example, goat F(ab′)2 anti-human IgG-alkaline phosphataseis a useful detectable secondary antibody that can be purchased fromJackson Immuno-Research (West Grove, Pa.).

[0064] A secondary antibody also can be rendered detectable by labelingwith a fluorochrome. Such a fluorochrome emits light of ultraviolet orvisible wavelength after excitation by light or another energy source.DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin,R-phycoerythrin, rhodamine, Texas red or lissamine, for example, is afluorochrome that can be linked to a secondary antibody and used todetect the presence or absence of a complex. A particularly usefulfluorochrome is fluorescein or rhodamine. Methods of conjugating andusing these and other suitable fluorochromes are described, for example,in Van Vunakis and Langone, Methods in Enzymology, Volume 74, Part C(1991), which is incorporated herein by reference. A secondary antibodylinked to a fluorochrome also can be obtained from commercial sources.For example, goat F(ab′)₂ anti-human IgG-FITC is available from TagoImmunologicals (Burlingame, Calif.).

[0065] A secondary antibody also can be labeled with a chemiluminescentmarker. Such a chemiluminescent secondary antibody is convenient forsensitive, non-radioactive detection of a complex containing an I-2polypeptide and can be obtained commercially from various sources suchas Amersham Lifesciences, Inc. (Arlington Heights, Ill.).

[0066] A secondary antibody further can be rendered detectable bylabeling with a radioisotope. An iodine-125 labeled secondary antibodyis a particularly useful detectable secondary antibody (see, forexample, Harlow and Lane, supra, 1988).

[0067] A signal from a detectable secondary antibody can be analyzed,for example, using a spectrophotometer to detect color from achromogenic substrate; a fluorometer to detect fluorescence in thepresence of light of a certain wavelength; or a radiation counter todetect radiation, such as a gamma counter for detection of iodine-125.For detection of an enzyme-linked secondary antibody, for example, aquantitative analysis can be made using a spectrophotometer such as anEMAX Microplate Reader (Molecular Devices, Menlo Park, Calif.) inaccordance with the manufacturer's instructions. If desired, the assaysof the invention can be automated or performed robotically, and thesignal from multiple samples can be detected simultaneously.

[0068] The assays of the present invention can be forward, reverse orsimultaneous as described in U.S. Pat. No. 4,376,110, issued Mar. 8,1983, to David et al., which is incorporated herein by reference. In theforward assay, each reagent is sequentially contacted with an I-2polypeptide of the invention. If desired, separation of bound fromunbound reagent can be performed before the addition of the nextreagent. In a reverse assay, all reagents are pre-mixed prior tocontacting with I-2 polypeptide. A modified reverse assay is describedin U.S. Pat. No. 4,778,751 issued Oct. 18, 1988, to El Shami et al.,which is incorporated herein by reference. In a simultaneous assay, allreagents are separately but contemporaneously contacted with an I-2polypeptide of the invention. A reagent is any component useful inperforming the assays of the present invention, for example, the sample,I-2 polypeptide, detectable secondary antibody, washing buffer or othersolutions.

[0069] Separation steps for the various assay formats described herein,including the removal of unbound secondary antibody from the complex,can be performed by methods known in the art (Harlow and Lane, supra,1988). For example, washing with a suitable buffer can be followed byfiltration, aspiration or magnetic separation. If the I-2 polypeptide oran immunoreactive fragment thereof is immobilized on a particulatesupport, such as on microparticles, the particulate material can becentrifuged, if desired, followed by removal of wash liquid. If the I-2polypeptide or an immunoreactive fragment thereof is immobilized on amembrane, filter or well, a vacuum or liquid absorbing apparatus can beapplied to the opposite side of the membrane, filter or well to draw thewash liquid away from the complex.

[0070] The invention also provides methods of determining susceptibilityto IBD in an individual by obtaining a sample from the individual;contacting the sample with an I-2 polypeptide, or immunoreactivefragment thereof, under conditions suitable to form a complex of the I-2polypeptide, or the immunoreactive fragment thereof, and antibody to theI-2 polypeptide; and detecting the presence or absence of the complex,where the presence of the complex indicates that the individual issusceptible to IBD.

[0071] The term “individual,” as used herein, means any animal capableof having inflammatory bowel disease, including a human, non-humanprimate, rabbit, rat or mouse, provided that the animal does not haveinflammatory bowel disease as defined by the clinical, endoscopic andhistopathologic parameters disclosed herein. A sample to be assayedaccording to the methods of the invention can be obtained from any suchindividual.

[0072] As used herein, the term “increased susceptibility to IBD” asindicated by the presence of a complex of I-2 polypeptide and antibodyto I-2 polypeptide, means a reduced ability to resist IBD-causingfactors, as compared with an individual from whom a sample is obtainedthat does not form a complex when contacted with I-2 polypeptide orimmunoreactive fragment thereof. Increased susceptibility to IBD in anindividual does not mean the individual will necessarily develop IBD.However, increased susceptibility to IBD in an individual is associatedwith an increased probability of having IBD in the future.

[0073] The term “immunoreactive fragment,” as used in reference to anI-2 polypeptide, means a peptide or polypeptide portion of an I-2polypeptide that has immunoreactivity as defined by the ability of ananti-I-2 antibody-positive sample to form a complex with the I-2polypeptide. Thus, the term “immunoreactive fragment of an I-2polypeptide” as used herein, means a peptide or polypeptide that has anamino acid sequence that is substantially the same as a portion of theamino acid sequence provided as SEQ ID NO: 2 and immunoreactivity asdefined by the ability to form a complex with an anti-I-2antibody-positive sample such as an I-2 reactive serum sample from aCrohn's disease patient. In general, an immunoreactive fragment has fromabout three amino acids to the full-length of an I-2 polypeptide. Animmunoreactive fragment of an I-2 polypeptide can have, for example, atleast 5, 8, 10, 12, 15, 18, 20 or 25 amino acids. For example, animmunoreactive fragment of an I-2 polypeptide can be from five to fiftyamino acids, from eight to fifty amino acids, or from ten to fifty aminoacids. More preferably, an immunoreactive fragment has from eight totwenty amino acids or from ten to twenty amino acids. Most preferably,an immunoreactive fragment has from twelve to twenty amino acids or fromfifteen to twenty amino acids.

[0074] An immunoreactive fragment of an I-2 polypeptide can beidentified by the ability to form a complex with an I-2 reactive sample,for example, an I-2 reactive CD patient serum sample. For example, animmunoreactive fragment of an I-2 polypeptide can be identified by itsability to form a complex when contacted with I-2 reactive CD sera.Assays for the formation of a complex between an antigen and anti-I-2serum sample are disclosed herein. An ELISA assay can be particularlyuseful in identifying an immunoreactive fragment of an I-2 polypeptide(see Example IIB).

[0075] Identification of a microbial sequence associated with IBDimplicates microbes in the pathogenesis of IBD and provides a valuablereagent for ameliorating inflammatory bowel disease. Furthermore, asdisclosed herein, the I-2 polypeptide can elicit a T cell response, asdemonstrated by the proliferation of murine T cells in response to aGST-I-2 fusion polypeptide (SEQ ID NO: 2), indicating that the I-2polypeptide antigen can contribute to the etiology of inflammatory boweldisease. Based on identification and isolation of the IBD-associated I-2sequence, there are provided methods of inducing tolerance in a patientwith IBD as well as methods of preventing IBD in a healthy individual.

[0076] Thus, the present invention provides methods of inducingtolerance in a patient with inflammatory bowel disease by administeringan effective dose of I-2 polypeptide, or tolerogenic fragment thereof,to the patient with IBD. The methods of the invention can beparticularly useful for treating a patient having Crohn's disease. In amethod of the invention for inducing tolerance, the I-2 polypeptide tobe administered can have, for example, the amino acid sequence of SEQ IDNO: 2.

[0077] As used herein, the term “patient with inflammatory boweldisease” means a patient having Crohn's disease or ulcerative colitis.

[0078] As used herein, the term “effective dose” means the amount of anI-2 polypeptide, or a tolerogenic fragment thereof, useful for inducingtolerance in a patient with IBD. For induction of oral tolerance, forexample, multiple smaller oral doses can be administered or a large dosecan be administered. Such doses can be extrapolated, for example, fromthe induction of tolerance in animal models (see, for example, Trenthamet al., Science 261:1727-1730 (1993), which is incorporated herein byreference).

[0079] An effective dose of an I-2 polypeptide or a tolerogenic fragmentthereof for inducing tolerance can be administered by methods well knownin the art. Oral tolerance is well-recognized in the art as a method oftreating autoimmune disease (see, for example, Weiner, HospitalPractice, pp. 53-58 (Sep. 15, 1995), which is incorporated herein byreference). For example, orally administered autoantigens suppressseveral experimental autoimmune models in a disease- andantigen-specific fashion; the diseases include experimental autoimmuneencephalomyelitis, uveitis, and myasthenia, collagen- andadjuvant-induced arthritis, and diabetes in the NOD mouse (see, forexample, Weiner et al., Ann. Rev. Immunol. 12:809-837 (1994), which isincorporated herein by reference). Furthermore, clinical trials of oraltolerance have produced positive results in treating multiple sclerosis,rheumatoid arthritis and uveitis. In addition, parenteral administrationof an I-2 polypeptide, or a tolerogenic fragment thereof, can be used toinduce tolerance. Subcutaneous injection, for example, can be used todeliver an I-2 polypeptide, or a tolerogenic fragment thereof, to an IBDpatient, for example, a patient having Crohn's disease (Johnson, Ann.Neurology 36(suppl.):S115-S117 (1994), which is incorporated herein byreference).

[0080] The term “tolerogenic fragment,” as used in reference to an I-2polypeptide of the invention, means a peptide or polypeptide portion ofthe polypeptide that has tolerogenic activity as defined by its abilityeither alone, or in combination with another molecule, to produce adecreased immunological response. Thus, a tolerogenic fragment of an I-2polypeptide is a peptide or polypeptide that has substantially the sameamino acid sequence as a portion of SEQ ID NO: 2 and tolerogenicactivity as defined by its ability either alone, or in combination withanother molecule, to produce a decreased immunological response. Atolerogenic fragment of an I-2 polypeptide can have from about threeamino acids to about 90 amino acids. A tolerogenic fragment of an I-2polypeptide can have, for example, at least 5, 8, 10, 12, 15, 18, 20 or25 amino acids. For example, a tolerogenic fragment of an I-2polypeptide can have from five to fifty amino acids, from eight to fiftyamino acids, or from ten to fifty amino acids. More preferably, atolerogenic fragment has from eight to twenty amino acids or from ten totwenty amino acids. Most preferably, a tolerogenic fragment has fromtwelve to twenty amino acids or from fifteen to twenty amino acids.

[0081] A tolerogenic fragment of an I-2 polypeptide can be identifiedusing a variety of assays, including in vitro assays such as T-cellproliferation or cytokine secretion assays and in vivo assays such asthe induction of tolerance in murine models of inflammatory boweldisease. T-cell proliferation assays, for example, are well recognizedin the art as predictive of tolerogenic activity (see, for example,Miyahara et al., Immunol. 86:110-115 (1995) or Lundin et al, J. Exp.Med. 178:187-196 (1993), each of which is incorporated herein byreference). A T-cell proliferation assay can be performed by culturingT-cells with irradiated antigen-presenting cells, such as normal spleencells, in microtiter wells for 3 days with varying concentrations of thefragment of an I-2 polypeptide to be assayed; adding ³H-thymidine; andmeasuring incorporation of ³H-thymidine into DNA. In such an assay, afragment of an I-2 polypeptide can be tested for activity, for example,at concentrations of 20 μg/ml and 40 μg/ml.

[0082] A tolerogenic fragment of an I-2 polypeptide can be identifiedusing a T-cell cytokine secretion assay known in the art. For example, Tcells can be cultured with irradiated antigen-presenting cells inmicrotiter wells with varying concentrations of the fragment of interestand, after three days, the culture supernatants can be assayed for IL-2,IL-4 or IFN-γ as described in Czerinsky et al., Immunol. Rev. 119:5-22(1991), which is incorporated herein by reference.

[0083] Primary T-cells for use in a T-cell proliferation assay orcytokine secretion assay, for example, can be isolated from laminapropria or peripheral blood. In addition, a convenient source of T-cellsfor use in an in vitro assay for tolerogenic activity can be a T-cellline established from an IBD patient such as a Crohn's disease patient,from a murine model of IBD or from a healthy animal immunized with anI-2 polypeptide of the invention. A preferred source of T-cells for usein identifying a tolerogenic fragment of an I-2 polypeptide is a Crohn'sdisease patient.

[0084] A T-cell line can be established from a patient with CD or UC,for example, by culturing T lymphocytes with about 1 μg/ml recombinantI-2 polypeptide or GST-I-2, in the presence of low concentrations ofgrowth-supporting IL-2 (about 10 μg/ml). A T-cell line can beestablished by culturing T lymphocytes with antigen-presenting cells andfeeding the cells on an alternating four to five day cycle with eitherIL-2 and I-2 polypeptide or IL-2 alone as described in Nanda et al., J.Exp. Med. 176:297-302 (1992), which is incorporated herein by reference.A cell line that develops into a reliably proliferating cell linedependent on the presence of I-2 polypeptide is particularly useful inidentifying tolerogenic fragments of I-2. The establishment of T-celllines from small intestinal mucosa is described, for example, in Lundinet al., supra, 1993. T cell lines dependent upon the presence of an I-2polypeptide and useful for identifying I-2 tolerogenic fragments can beprepared similarly.

[0085] A tolerogenic fragment of an I-2 polypeptide also can beidentified by its ability to induce tolerance in vivo, as indicated by adecreased immunological response, which can be a decreased T-cellresponse, such as a decreased proliferative response or cytokinesecretion response as described above, or a decreased antibody titer tothe antigen. A neonatal or adult mouse can be tolerized with a fragmentof an I-2 polypeptide, for example, and a T-cell response or anti-I-2polypeptide antibody titer can be assayed after challenging byimmunization. For example, a neonatal mouse can be tolerized within 48hours of birth by intraperitoneal administration of about 100 μg of afragment of an I-2 polypeptide emulsified with incomplete Freund'sadjuvant and subsequently immunized with I-2 polypeptide at about 8weeks of age (see, for example, Miyahara, supra, 1995). An adult mousecan be tolerized intravenously with about 0.33 mg of a fragment of anI-2 polypeptide, administered daily for three days (total dose 1 mg),and immunized one week later with an I-2 polypeptide. A decreased T-cellresponse, such as decreased proliferation or cytokine secretion, whichindicates tolerogenic activity, can be measured using T-cells harvested10 days after immunization. In addition, a decreased anti-I-2polypeptide antibody titer, which also indicates tolerogenic activity,can be assayed using blood harvested 4-8 weeks after immunization.Methods for assaying a T-cell response or anti-I-2 polypeptide antibodytiter are described above and are well known in the art.

[0086] A tolerogenic fragment of an I-2 polypeptide also can beidentified using a murine model of inflammatory bowel disease. Neonatalor adult mice having IBD-like disease can be tolerized with a fragmentof an I-2 polypeptide as described above, and the T-cell response oranti-I-2 polypeptide antibody titer assayed. A decreased T-cell responseor decreased antibody titer to the antigen indicates a decreasedimmunological response and, thus, serves to identify a tolerogenicfragment of an I-2 polypeptide. In addition, a tolerogenic fragment ofan I-2 polypeptide can be identified by the ability to reduce thefrequency, time of onset or severity of colitis in a murine model ofIBD.

[0087] Several well-accepted murine models of inflammatory bowel diseasecan be useful in identifying a tolerogenic fragment of an I-2polypeptide of the invention. For example, mice with target disruptionof the genes encoding the alpha subunit of the G-protein Gi2, are a wellknown model exhibiting features of human bowel disease (Hornquist etal., J. Immunol. 158:1068-1077 (1997); Rudolph et al., Nat. Genet.10:143-150 (1995), each of which is incorporated herein by reference).Mice deficient in IL-10 as described in Kuhn et al., Cell 75:263-274(1993), which is incorporated herein by reference, and mice deficient inIL-2 as described in Sadlack et al., Cell 75:253-261 (1993), which isincorporated herein by reference, also have colitis like disease and areuseful in identifying a tolerogenic fragment of an I-2 polypeptide ofthe invention. Furthermore, mice with mutations in T cell receptor (TCR)α, TCR β, TCR β×δ, or the class II major histocompatiblility complex(MHC) as described in Mombaerts et al., Cell 75:275-282 (1993), which isincorporated herein by reference, develop inflammatory bowel diseaseand, thus, are useful in identifying a tolerogenic fragment of an I-2polypeptide. Similarly, a fragment can be assayed for tolerogenicactivity in a SCID mouse reconstituted with CD45RB CD4+ T-cells, whichis a well-accepted model of inflammatory bowel disease, as described inPowrie et al., Immunity 1:553-562 (1994), which is incorporated hereinby reference. Additional animal models of IBD also are well known in theart (see, for example, Podolsky, Acta Gastroenterol. Belq. 60:163-165(1997); and Bregenholt et al., APMIS 105: 655-662 (1997), each of whichis incorporated herein by reference). Thus, a tolerogenic fragment of anI-2 polypeptide can be readily identified by an in vitro or in vivoassay disclosed herein or by another assay well known in the art.

[0088] An immunoreactive or tolerogenic fragment of an I-2 polypeptidecan be identified by screening a large collection, or library, ofpeptides of interest or random peptides for immunoreactivity ortolerogenic activity. For example, a panel of peptides spanning theentire sequence of an I-2 polypeptide can be screened forimmunoreactivity or tolerogenic activity as described above. Such apanel can be a panel of 15-mer peptides spanning the sequence of the I-2polypeptide (SEQ ID NO: 2), each overlapping by three or five residueshifts using the Mimotope cleavable pin technology (Cambridge ResearchBiochemicals, Wilmington, Del.), as described by Geysen et al., Science235:1184 (1987), which is incorporated herein by reference. The panel issubsequently screened for immunoreactivity or tolerogenic activity usingone of the assays described above (see, for example, Miyahara et al.,supra, 1995, which is incorporated herein by reference). A library ofpeptides to be screened also can be a population of peptides related inamino acid sequence to SEQ ID NO: 2 but having one or more amino acidsthat differ from SEQ ID NO: 2.

[0089] Additional peptides to be screened include, for example, taggedchemical libraries of peptides and peptidomimetic molecules. Peptidelibraries also comprise those generated by phage display technology.Phage display technology includes the expression of peptide molecules onthe surface of phage as well as other methodologies by which a proteinligand is or can be associated with the nucleic acid which encodes it.Methods for production of phage display libraries, including vectors andmethods of diversifying the population of peptides which are expressed,are well known in the art (see, for example, Smith and Scott, MethodsEnzymol. 217:228-257 (1993); Scott and Smith, Science 249:386-390(1990); and Huse, WO 91/07141 and WO 91/07149, each of which isincorporated herein by reference). These or other well known methods canbe used to produce a phage display library which can be screened, forexample, with one of the disclosed assays for immunoreactivity ortolerogenic activity. If desired, a population of peptides can beassayed for activity en masse. For example, to identify animmunoreactive fragment of an I-2 polypeptide, a population of peptidescan be assayed for the ability to form a complex with a samplecontaining anti-I-2 polypeptide reactivity; the active population can besubdivided and the assay repeated in order to isolate the immunoreactivefragment from the population.

[0090] An immunoreactive or tolerogenic fragment of an I-2 polypeptidealso can be identified by screening, for example, fragments of thepolypeptide produced by chemical or proteolytic cleavage. Methods forchemical and proteolytic cleavage and for purification of the resultantprotein fragments are well known in the art (see, for example,Deutscher, Methods in Enzvmology, Vol. 182, ” Guide to ProteinPurification,” San Diego: Academic Press, Inc. (1990), which isincorporated herein by reference). For example, a chemical such ascyanogen bromide or a protease such as trypsin, chymotrypsin, V8protease, endoproteinase Lys-C, endoproteinase Arg-C or endoproteinaseAsp-N can be used to produce convenient fragments of an I-2 polypeptidethat can be screened for immunoreactivity or tolerogenic activity usingone of the assays disclosed herein.

[0091] As used herein, the term “fragment” means a peptide, polypeptideor compound containing naturally occurring amino acids, non-naturallyoccurring amino acids or chemically modified amino acids. Animmunoreactive or tolerogenic fragment of an I-2 polypeptide also can bea peptide mimetic, which is a non-amino acid chemical structure thatmimics the structure of a peptide having an amino acid sequence,provided that the peptidomimetic retains immunoreactivity or tolerogenicactivity, as defined herein Such a mimetic generally is characterized asexhibiting similar physical characteristics such as size, charge orhydrophobicity in the same spatial arrangement found in its peptidecounterpart. A specific example of a peptide mimetic is a compound inwhich the amide bond between one or more of the amino acids is replaced,for example, by a carbon-carbon bond or other bond well known in the art(see, for example, Sawyer, Peptide Based Drug Design, ACS, Washington(1995), which is incorporated herein by reference).

[0092] As used herein, the term “amino acid” refers to one of the twentynaturally occurring amino acids, including, unless stated otherwise,L-amino acids and D-amino acids. The term amino acid also refers tocompounds such as chemically modified amino acids including amino acidanalogs, naturally occurring amino acids that are not usuallyincorporated into proteins such as norleucine, and chemicallysynthesized compounds having properties known in the art to becharacteristic of an amino acid, provided that the compound can besubstituted within a peptide such that it retains immunoreactivity ortolerogenic activity. Examples of amino acids and amino acids analogsare listed in Gross and Meienhofer, The Peptides: Analysis, Synthesis,Biology, Academic Press, Inc., New York (1983), which is incorporatedherein by reference. An amino acid also can be an amino acid mimetic,which is a structure that exhibits substantially the same spatialarrangement of functional groups as an amino acid but does notnecessarily have both the α-amino and α-carboxyl groups characteristicof an amino acid.

[0093] An immunoreactive or tolerogenic fragment of an I-2 polypeptidecan be produced or synthesized using methods well known in the art. Suchmethods include recombinant DNA methods and chemical synthesis methodsfor production of a peptide. Recombinant methods of producing a peptidethrough expression of a nucleic acid sequence encoding the peptide in asuitable host cell are well known in the art and are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd, Vols 1 to 3, Cold Spring Harbor Laboratory Press, New York (1989),which is incorporated herein by reference. The sequence of a nucleicacid molecule encoding an I-2 polypeptide is disclosed herein as SEQ IDNO: 1.

[0094] An immunoreactive or tolerogenic fragment of an I-2 polypeptidealso can be produced by chemical synthesis, for example, by the solidphase peptide synthesis method of Merrifield et al., J. Am. Chem. Soc.85:2149 (1964), which is incorporated herein by reference. Standardsolution methods well known in the art also can be used to synthesize animmunoreactive or tolerogenic fragment useful in the invention (see, forexample, Bodanszky, Principles of Peptide Synthesis, Springer-Verlag,Berlin (1984) and Bodanszky, Peptide Chemistry, Springer-Verlag, Berlin(1993), each of which is incorporated herein by reference). A newlysynthesized peptide can be purified, for example, by high performanceliquid chromatography (HPLC), and can be characterized using, forexample, mass spectrometry or amino acid sequence analysis.

[0095] The present invention also provides tolerogenic compositions thatcontain an I-2 polypeptide and are useful in inducing tolerance in apatient with IBD. In particular, the invention provides compositionsincluding an I-2 polypeptide having substantially the same amino acidsequence as SEQ ID NO: 2, or tolerogenic fragment thereof, combined witha tolerogizing molecule.

[0096] In a composition of the invention, the I-2 polypeptide can have,for example, the amino acid sequence SEQ ID NO: 2. A tolerogenicfragment useful in a composition of the invention can have, for example,at least ten contiguous amino acids of SEQ ID NO: 2.

[0097] Various molecules are known in the art to cause, promote orenhance tolerance. See, for example, U.S. Pat. No. 5,268,454, andcitations therein, which are incorporated herein by reference. As usedherein, the term “tolerogizing molecule” means a molecule, compound orpolymer that causes, promotes or enhances tolerogenic activity whencombined with an I-2 polypeptide of the invention, or fragment thereof.A tolerogizing molecule can be, for example, conjugated to an I-2polypeptide. Such tolerogizing molecules include, for example,polyethylene glycol and are well known in the art (see, for example,U.S. Pat. No. 5,268,454, supra).

[0098] The invention also provides methods of preventing IBD in anindividual by administering an effective dose of an I-2 polypeptide, ortolerogenic fragment thereof, to the individual. The methods of theinvention are particularly useful for preventing IBD, for example,Crohn's disease in an individual having increased susceptibility to IBD.Such methods can be particularly useful for preventing IBD when aneffective dose of the antigen or tolerogenic fragment is administered toa newborn individual.

[0099] Enteric bacteria have been shown to play a role in thepathogenesis of several diseases. For example, H. pylori has beenimplicated in pathogenesis of peptic ulcer disease, and antibioticsagainst H. pylori can be used to effectively treat this disease (see,for example, Sontag, Am. J. Gastroenterol. 92:1255-1261 (1997); andPipkin et al., Helicobactor. 2:159-171 (1997), each of which isincorporated herein by reference). In Crohn's disease, intestinalinflammation is notable for its frequent responsiveness to antibioticsand susceptibility to bacterial fecal flow (Gui et al., J. Antimicrob.Chemother. 39:393-400 (1997); Prantera et al., Am. J. Gastroenterol.91:328-332 (1996); and Janowitz et al., Inflamm. Bowel. Dis. 4:29-39(1998), each of which is incorporated herein by reference). Commonintestinal colonists and novel pathogens have been implicated in CD bydirect detection or by disease-associated anti-microbial immuneresponses (Blaser et al., Gastroenterology 87:888-894 (1984); Elsaghieret al., Clin. Exp. Immunol. 90:503-508 (1992); Del Prete et al., J.Microbiol. Methods 33:105-114 (1998); Metcalf, British Medical Journal316:166 (1998), each of which is incorporated herein by reference; andSendid et al., supra, 1996). Furthermore, in most animal models ofchronic colitis, lumenal micro-organisms are a necessary co-factor fordisease. Dietary antigens also have been implicated in IBD pathogenesis(Sonnenberg, Gut 31:1037-1040 (1990); Davidson et al., Clin. Exp.Immunol. 35:147-148 (1979); and Knoflach et al., Gastroenterology92:479-485 (1987), each of which is incorporated herein by reference).Despite extensive research, the pathogenic organism remains to beidentified.

[0100] As disclosed herein, colonic microbes harbored in inflamedlesions in Crohn's disease patients contain a nucleic acid sequenceencoding the I-2 polypeptide antigen (SEQ ID NO: 2). Based on thisfinding, the microbial organism can be isolated and used for discoveryof agents that reduce the viability or growth of the organism, therebydiminishing the immune stimulus contributing to Crohn's and amelioratingsymptoms of the disease.

[0101] Thus, the invention provides a method of identifying an agentuseful in treating inflammatory bowel disease. The method includes thesteps of obtaining a specimen of an enteric microbe from a patient withinflammatory bowel disease; isolating from the specimen a microbialspecies that includes a nucleic acid molecule encoding an I-2polypeptide; contacting the microbial species with an agent; andassaying for reduced growth or viability of the microbial species ascompared to the growth or viability in the absence of the agent, wherethe reduced growth or viability of the microbial species indicates thatthe agent is an agent useful in treating inflammatory bowel disease. Themethods of the invention can be particularly useful for screening agentswhich are antibiotics and for identifying agents for the treatment ofCrohn's disease. One skilled in the art understands that the microbialspecies which is contacted with an agent in the methods of the inventioncan be a single microbial species or can be a mixture of two or moremicrobial species, where at least one species contains a nucleic acidsequence encoding an I-2 polypeptide.

[0102] Isolation of a microbial species that includes a nucleic acidmolecule encoding an I-2 polypeptide can be performed by culturing theI-2 positive specimen on a variety of mediums and under aerobic andanaerobic conditions as described in Example IV; isolates aresubsequently screened for the presence of the I-2 sequence using, forexample, PCR analysis. Exemplary culture conditions are set forth inFIG. 7.

[0103] As used herein, the term “agent” means a biological or chemicalcompound such as a simple or complex organic molecule, a peptide, aprotein, an antibody, a lipid or an oligonucleotide.

[0104] An agent identified by the methods of the invention reduces theviability or growth of a microbial species that contains a nucleic acidmolecule encoding an I-2 polypeptide. Thus, an agent useful in treatingIBD can be an agent that functions to kill or slow the growth of amicrobial species that contains a nucleic acid molecule encoding an I-2polypeptide. An agent useful in treating IBD can be a bacteriostatic orbacteriocidal agent such as a bacterial antibiotic, which is a moleculethat is produced by a microorganism or a plant, or a chemical derivativeof such a molecule, that can reduce the growth or viability of abacterial species that contains a nucleic acid molecule encoding an I-2polypeptide. One skilled in the art understands that an agent useful intreating IBD can function by a variety of mechanisms, for example, byinhibiting microbial protein synthesis, inhibiting microbial DNAsynthesis, inhibiting microbial cell wall synthesis or inhibitingsynthesis of an essential nutrient of a microbial species that containsa nucleic acid molecule encoding an I-2 polypeptide. Such an agent canselectively reduce the viability or growth of a particular microbialspecies that contains a nucleic acid molecule encoding an I-2polypeptide. An agent useful in treating IBD also can have activity inreducing the growth or viability of a broad spectrum of microbes. Oneskilled in the art understands that, preferably, an agent useful intreating IBD reduces the growth or viability of a microbial species thatcontains a nucleic acid molecule encoding an I-2 polypeptide withoutsignificantly altering the growth or viability of mammalian cells,especially human cells.

[0105] As used herein, the term “agent useful in treating IBD” means anagent that reduces the severity, frequency, or time of onset of one ormore symptoms of inflammatory bowel disease.

[0106] Although animal models of inflammatory bowel disease are known,these models do not involve an antigen associated with human IBD. Basedon the disclosed isolation of the IBD-associated I-2 polypeptide antigen(SEQ ID NO: 2) and the demonstration that murine T cells are responsiveto this antigen (see Example III), the invention provides novel animalmodels for IBD, in which disease is initiated with the I-2 antigenassociated with human disease. Thus, superior IBD animal models areprovided for identifying new IBD therapeutics, including antibiotics,anti-inflammatories and other drugs.

[0107] In particular, the invention provides a method of identifying anagent useful in treating inflammatory bowel disease by administering anI-2 polypeptide to a non-human animal, whereby one or symptoms of IBDare exhibited; administering an agent to the non-human animal; andassaying the level of the one or more symptoms characteristic of IBD,where a reduction in the level of the one or more symptoms as comparedto a control level indicates that the agent is an agent useful intreating IBD. The methods of the invention can be applied, for example,to identification of agents useful in treating Crohn's disease. The I-2polypeptide administered can have, for example, the amino acid sequenceSEQ ID NO: 2. A non-human animal particularly useful in the methods ofthe invention can be, for example, a mouse deficient in Gai2, TCRα orIL-10.

[0108] As set forth above, the term “agent” means a biological orchemical compound such as a simple or complex organic molecule, apeptide, a protein, an antibody, a lipid or an oligonucleotide. An agentcan be, for example, an antibiotic or anti-inflammatory compound.

[0109] The methods of the invention rely on the use of a non-humananimal, which can exhibit one or more symptoms of inflammatory boweldisease in response to an antigenic trigger. Any non-human animal modelof disease can be useful in the methods of the invention includingnon-human primate, rat or mouse model of IBD. A variety of mouse modelsof IBD well known in the art can be particularly useful in the invention(see, for example, Podolsky, supra, 1997; Bregenholt et al., supra,1997). Such mouse models include those described hereinabove, forexample, mice deficient in Gαi2 (Gαi2 −/−; Rudolph et al., supra, 1995);mice deficient in IL-10 (IL-10 −/−; Kuhn et al., supra, 1993); micedeficient in TCRα (TCRα −/−); mice deficient in TCRβ (TCRβ −/−); micedeficient in keratin 8; mice deficient in IL-2 (IL-2 −/−; Sadlack,supra, 1993); and SCID mice reconstituted with CD45RB CD4+ T cells(Powrie et al., supra, 1994). Non-human animal models useful in themethods of the invention also include animals expressing transgenes forhuman HLA-B27 (with β2-microglobulin) or animals expressing a dominantnegative construct that functionally blocks N-cadherin (Podolsky, supra,1997).

[0110] One skilled in the art understands that, in order to initiate oneor more symptoms of IBD in a non-human animal model, an 1-2 polypeptideof the invention can be administered by a variety of routes. Preferredadministration of an I-2 polypeptide to initiate one or more symptoms ofIBD in a non-human animal model of the disease is by enemaadministration or by administering a cellular composition in which theI-2 polypeptide is expressed using E. coli.

[0111] Local administration of free antigen can be achieved by enema,for example, as described in Mahler et al., Am. J. Physiol.274:G544-G551 (1998). An I-2 polypeptide also can be administered bycolonization of the mouse colon with antigen-bearing microorganisms, forexample, E. coli (Cahill et al., Infect Immuno., 65:3126-3131 (1997)) oranother bacterial species (Kullberg et al., Infect. Immun. 66:5157-5166(1998); Li et al., Infect. Immun. 66:5477-5484 (1998); and Cahill,supra, 1997). In a non-human animal model of IBD, one or more symptomsof IBD also can be induced using a T cell line reactive with a bacterialantigen, where the T cell line is derived from a colitis susceptiblemouse strain. Such a T cell line can be, for example, an E. colireactive T cell line produced from C3H/HeJBir mice (Cong et al., J. Exp.Med. 187:855-864 (1998)). A C.B-17Scid mouse also can be colonized withantigen-bearing bacteria, either naturally or by intra-intestinalinfection (Kullberg et al., supra, 1998; Li et al., supra, 1998; andCong et al., supra, 1998). Transfer of a T cell line into such a mouseresults in a robust antigen-specific inflammatory bowel disease model(Kullberg et al., supra, 1998; and Cong et al., supra, 1998).

[0112] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Identification of the I-2 and I-1 Target Antigens

[0113] This example describes isolation of novel microbial nucleic acidsequences encoding the I-1 and I-2 polypeptides.

[0114] A. Representational Difference Analysis

[0115] Representational difference analysis (RDA), which uses PCR drivensubtractive cloning to identify DNA sequences found preferentially in aninfected area, was utilized to examine sequences differentially presentin involved versus uninvolved CD mucosa.

[0116] RDA was performed essentially as described in Chang et al.,Science 266:1865-1869 (1994), which is incorporated herein by reference.Briefly, mononuclear cells from the lamina propria of a Crohn's diseasepatient were isolated from an area with ulcerations (tester cells) andfrom an area macroscopically free of disease (driver cells), and DNApurified from each cell population. Using RDA, the DNA sequences fromthe area free of disease (driver DNA) were subtracted from the DNA foundat the site of ulceration (tester DNA), leaving sequences preferentiallyfound in the inflamed area.

[0117] Four sequences were obtained, two of which were human sequences.The remaining two sequences, designated I-1 and I-2, showed nosignificant homology to any human genes. The I-2 nucleic acid sequence(SEQ ID NO: 1) and encoded polypeptide (SEQ ID NO: 2) are shown in FIG.1A. The I-1 nucleic acid sequence (SEQ ID NO: 3) and two encoded openreading frames (SEQ ID NOS: 4 and 5) are shown in FIG. 1B.

[0118] B. PCR Analysis of Archived Paraffin Embedded Tissue Samples

[0119] Paraffin embedded tissue samples from CD patients, UC patients,or non-IBD controls were subject to PCR analysis. Briefly, sample wasamplified with the I-2 specific primers 5′-CCGTGGGCATCCAGTCCG-3′ (SEQ IDNO: 9) and 5′-TCTGCTCATACACGTCACG-3′ (SEQ ID NO: 10) using standard PCRconditions with a final concentration of 4 mM MgCl₂. In particular, thereactions consisted of 26.75 μl H₂O; 5.0 μl 10× PCR Buffer; 5.0 μl 25 mMMgCl₂; 1.0 μl 10 mM each dNTP; 1.0 μl each of SEQ ID NOS: 9 and 10; and0.25 μl Taq polymerase (5 units/μl). The reactions were incubated at 94°C. for 5 minutes; followed by 39 cycles of 94° C. for 30 seconds/65° C.for 30 seconds and 72° C. for 30 seconds, followed by a final 5 minuteextension at 72° C. Reactions were analyzed on 2% agarose, with thepresence of the I-2 sequence indicated by the expected 285 bp fragment.

[0120] As shown in FIG. 3, the I-2 sequence (SEQ ID NO: 1) was presentsignificantly more frequently in involved CD samples than in UC samplesor non-IBD samples. These results indicate that the I-2 polypeptideantigen (SEQ ID NO: 2) is associated with inflammatory bowel disease, inparticular, with Crohn's disease.

EXAMPLE II Differential Reactivity of IBD Patient And Normal Sera to theI-2 Polypeptide

[0121] This example demonstrates that the I-2 polypeptide isdifferentially reactive with Crohn's disease patient sera as compared tonormal sera.

[0122] A. GST-I-2 Fusion Protein

[0123] The full-length I-2 encoding nucleic acid sequence (SEQ ID NO: 1)was cloned into the GST expression vector pGEX. After expression in E.coli, the protein was purified on a GST column. The purified protein wasshown to be of the expected molecular weight by silver staining, and hadanti-GST reactivity upon western analysis.

[0124] B. ELISA Analysis

[0125] ELISA analysis was performed with GST-I-2 (SEQ ID NO: 2) fusionpolypeptide using diluted patient or normal serum. Reactivity wasdetermined after subtracting reactivity to GST alone. Varying dilutionsof CD sera and sera from normal individuals were assayed for IgGreactivity to the GST-I-2 fusion polypeptide. As shown in FIG. 4A,dilutions of 1:100 to 1:1000 resulted in significantly higher anti-I-2polypeptide reactivity for the CD sera as compared to normal sera. Theseresults indicate that the I-2 polypeptide (SEQ ID NO: 2) isdifferentially reactive with Crohn's disease sera as compared to normalsera.

[0126] Serum IgA reactivity of UC, CD and normal sera to the I-2polypeptide (SEQ ID NO: 2) was assayed as described below. As shown inFIG. 4B, using a cutoff that is two standard deviations above the meanvalue for the normal population, nine of ten CD values were positive,while none of the normal serum samples were positive. Furthermore, sevenof ten Crohn's disease patients showed an OD₄₀₅ greater than 0.3, whilenone of the UC or normal samples were positive by this measure. Theseresults indicate that immunoreactivity to the I-2 polypeptide, inparticular, IgA immunoreactivity, can be used to diagnose Crohn'sdisease.

[0127] Human IgA and IgG antibodies that bind the I-2 polypeptide (SEQID NO: 2) were detected by direct ELISA assays essentially as follows.Plates (Immulon 3; DYNEX Technologies; Chantilly, Va.) were coatedovernight at 4° C. with 100 μl/well GST-I-2 fusion polypeptide (5 μg/mlin borate buffered saline, pH 8.5). After three washes in 0.05% Tween 20in phosphate buffered saline (PBS), the plates were blocked with 150μl/well of 0.5% bovine serum albumin in PBS, pH 7.4 (BSA-PBS) for 30minutes at room temperature. The blocking solution was then replacedwith 100 μl/well of Crohn's disease serum, ulcerative colitis serum, ornormal control serum, diluted 1:100. The plates were then incubated for2 hours at room temperature and washed as before. Alkaline phosphataseconjugated secondary antibody [goat anti-human IgA (α-chain specific),Jackson ImmunoResearch, West Grove, Pa.] was added to the IgA plates ata dilution of 1:1000 in BSA-PBS. For IgG reactivity, alkalinephosphatase conjugated secondary antibody (goat anti-human IgG (γ-chainspecific), Jackson ImmunoResearch) was added. The plates were incubatedfor 2 hours at room temperature before washing three times with 0.05%Tween 20/PBS followed by another three washes with Tris buffered normalsaline, pH 7.5. Substrate solution (1.5 mg/ml disodium P-nitrophenolphosphate (Aresco; Solon, Ohio) in 2.5 mM MgCl₂, 0.01 M Tris, pH 8.6)was added at 100 μl/well, and color allowed to develop for one hour. Theplates were then analyzed at 405 nm.

[0128] C. Histological Analysis

[0129] Rabbit anti-I-2 antibodies are prepared using purified GST-I-2fusion protein as the immunogen. GST binding antibodies are removed byadherence to GST bound to an agarose support (Pierce), and the rabbitsera validated for anti-I-2 immunoreactivity by ELISA analysis.

[0130] Slides are prepared from paraffin embedded biopsy specimens fromCD, UC and normal controls. Hematoxylin and eosin staining areperformed, followed by incubation with I-2 specific antiserum. Bindingof antibodies is detected with peroxidase labeled anti-rabbit secondaryantibodies (Pierce). The assay is optimized to maximize the signal tobackground and the distinction between CD and control populations.

EXAMPLE III Reactivity of CD4+T Cells with the I-2 Polypeptide

[0131] This example demonstrates that T cells derived from normal miceproliferate in response to the I-2 antigen (SEQ ID NO: 2).

[0132] CD4+ T cells were isolated from normal C57BL/6J mice, and T cellproliferation assayed in response to the I-2 antigen as follows. Toprepare the CD4+ T cells, spleens from 8-10 week old female mice wereremoved and placed into cell suspension. The cells were depleted for Bcells and antigen presenting cells (APCs) using nylon wool and thendepleted for CD8+T cells using anti-CD8 magnetic beads. Flow cytometrywas used to determine the purity of the CD4+ T cell suspension.

[0133] For preparing APCs, spleens from 8-10 week old females areremoved, placed into cell suspension, and pulsed overnight with 0, 2,10, or 15 μg/ml of the I-2 polypeptide. The APCs are irradiated with3,000 rads before being added to the T cell cultures. To assay forantigen specific proliferation of T cells, 4×10⁵ CD4+ T cells/well wereincubated with 4×10⁵ antigen-pulsed APCs/well in a 96-well flat bottomedtissue culture plate at 37 C. in 5% CO₂ humidified air. After varyingperiods of incubation, 0.5 μCi of [³H]-thymidine was added to eachculture for the last 18 hours of incubation. The cells were harvestedand proliferation assessed as the amount of [³H]-thymidine into cell DNAby scintillation counting.

[0134] As shown in FIG. 5, the I-2 polypeptide antigen (SEQ ID NO: 2)stimulates T cell proliferation at concentrations of 5 μg/ml and 25μg/ml. These results demonstrate that the I-2 polypeptide (SEQ ID NO: 2)associated with human inflammatory bowel disease can elicit a T cellresponse by murine cells and indicate that an I-2 polypeptide cancontribute to the etiology of inflammatory bowel disease.

[0135] B. Cytokine Expression in Response to the 1-2 Polypeptide

[0136] A role for regulatory T cells is supported by studies withCD45RB^(low), CD28+CD45RB^(low) and CD25+CD45RB^(low) cells. Cytokineexpression was therefore analyzed in response to the I-2 polypeptide. Tcell proliferation cultures were used for cytokine assays. On day 4 ofthe T cell proliferation assay, supernatants were analyzed for IL-10 andinterferon-y expression. As shown in FIG. 6, expression of interferon-y,a Th1 cytokine regulator, is induced by the GST-I-2 polypeptide (SEQ IDNO: 2). Furthermore, FIG. 6 shows that IL-10 expression, which can actto down regulate a Th1 cytokine response, is induced by exposure of CD4+T cells to as little at 2 μg/ml GST-I-2 polypeptide (SEQ ID NO: 2).These results indicate that an animal model of IBD in which disease isinitiated by the I-2 polypeptide antigen (SEQ ID NO: 2) can be a usefulmodel of human disease.

EXAMPLE IV Isolation and Identification of the Organism Containing theI-2 Seouence

[0137] This example describes isolation and identification of theorganism containing the I-2 sequence.

[0138] Tissue resections from CD patients are obtained from Cedars-Sinaiunder anaerobic conditions. DNA is extracted from a small piece oftissue using the Qiagen tissue prep kit, and tested for the presence ofthe I-2 nucleic acid sequence SEQ ID NO: 1 using the PCR assay describedabove. Cells from positive scoring sections are cultured under a varietyof conditions (see FIG. 7), and the isolates catalogued.

[0139] After assaying the isolates by PCR for 16S RNA to assure that thetarget DNA is accessible to amplification, isolates are assayed by PCRfor the I-2 nucleic acid sequence (SEQ ID NO: 1) as described in ExampleI. The 16S RNA of isolates containing the I-2 nucleic acid are sequencedto identify the organism essentially as described in Wilson andBlitchington, Applied and Environ. Microbiol. 62:2273-2278 (1996), whichis incorporated herein by reference.

[0140] All journal article, reference and patent citations providedabove, in parentheses or otherwise, whether previously stated or not,are incorporated herein by reference in their entirety.

[0141] Although the invention has been described with reference to theexamples above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 10 1 302 DNA Unknown Microbial Organism from the human gut 1 a gat ctggcc agc gcc gtg ggc atc cag tcc ggc agc atc ttt cat cac 49 Asp Leu AlaSer Ala Val Gly Ile Gln Ser Gly Ser Ile Phe His His 1 5 10 15 ttc aagagc aag gat gag ata ttg cgt gcc gtg atg gag gaa acc atc 97 Phe Lys SerLys Asp Glu Ile Leu Arg Ala Val Met Glu Glu Thr Ile 20 25 30 cat tac aacacc gcg atg atg cgc gct tca ctg gag gag gcg agc acg 145 His Tyr Asn ThrAla Met Met Arg Ala Ser Leu Glu Glu Ala Ser Thr 35 40 45 gtg cgc gaa cgcgtg ctg gcg ctg atc cgc tgc gag ttg cag tcg atc 193 Val Arg Glu Arg ValLeu Ala Leu Ile Arg Cys Glu Leu Gln Ser Ile 50 55 60 atg ggc ggc agt ggcgag gcc atg gcg gtg ctg gtc tac gaa tgg cgc 241 Met Gly Gly Ser Gly GluAla Met Ala Val Leu Val Tyr Glu Trp Arg 65 70 75 80 tcg ctg tcg gcc gaaggc cag gcg cac gtg ctg gcc ctg cgt gac gtg 289 Ser Leu Ser Ala Glu GlyGln Ala His Val Leu Ala Leu Arg Asp Val 85 90 95 tat gag cag atc t 302Tyr Glu Gln Ile 100 2 100 PRT Unknown Microbial organism from the humangut 2 Asp Leu Ala Ser Ala Val Gly Ile Gln Ser Gly Ser Ile Phe His His 15 10 15 Phe Lys Ser Lys Asp Glu Ile Leu Arg Ala Val Met Glu Glu Thr Ile20 25 30 His Tyr Asn Thr Ala Met Met Arg Ala Ser Leu Glu Glu Ala Ser Thr35 40 45 Val Arg Glu Arg Val Leu Ala Leu Ile Arg Cys Glu Leu Gln Ser Ile50 55 60 Met Gly Gly Ser Gly Glu Ala Met Ala Val Leu Val Tyr Glu Trp Arg65 70 75 80 Ser Leu Ser Ala Glu Gly Gln Ala His Val Leu Ala Leu Arg AspVal 85 90 95 Tyr Glu Gln Ile 100 3 392 DNA Unknown Microbial Organismfrom the human gut 3 a gat ctt gag cgt cat gag tgc ctg ggg tac gcc ttttca tcg cgt ccg 49 Asp Leu Glu Arg His Glu Cys Leu Gly Tyr Ala Phe SerSer Arg Pro 1 5 10 15 gcg gat cga gag tgg gtg ttt ttt cag ggc acg gtttcc tac aag gta 97 Ala Asp Arg Glu Trp Val Phe Phe Gln Gly Thr Val SerTyr Lys Val 20 25 30 cga gtg gcc agc cgt ttg ctc atc aat gaa agc cgg gcattg atg tcg 145 Arg Val Ala Ser Arg Leu Leu Ile Asn Glu Ser Arg Ala LeuMet Ser 35 40 45 gcg gca ttg gat ggt ttt ggc ata gtg ctc ggc ccg caa gacttc ctg 193 Ala Ala Leu Asp Gly Phe Gly Ile Val Leu Gly Pro Gln Asp PheLeu 50 55 60 cga acg gcg ttg gcg agt ggc gag ttg gtg cgg gtg ttg ccg gagttt 241 Arg Thr Ala Leu Ala Ser Gly Glu Leu Val Arg Val Leu Pro Glu Phe65 70 75 80 gag gct ccg agt cgg tcg atg cat ttg gtc tac acc gca aac cgccag 289 Glu Ala Pro Ser Arg Ser Met His Leu Val Tyr Thr Ala Asn Arg Gln85 90 95 cgt acc gcc aag ttg cgc tgc ttt gtc gag act gtg ctg gga cgt ttt337 Arg Thr Ala Lys Leu Arg Cys Phe Val Glu Thr Val Leu Gly Arg Phe 100105 110 ggt ccg gta tgaaggagca ccaccgtggc ggtcgccggg angcacctaa 386 GlyPro Val 115 agatct 392 4 115 PRT Unknown Microbial organism from thehuman gut 4 Asp Leu Glu Arg His Glu Cys Leu Gly Tyr Ala Phe Ser Ser ArgPro 1 5 10 15 Ala Asp Arg Glu Trp Val Phe Phe Gln Gly Thr Val Ser TyrLys Val 20 25 30 Arg Val Ala Ser Arg Leu Leu Ile Asn Glu Ser Arg Ala LeuMet Ser 35 40 45 Ala Ala Leu Asp Gly Phe Gly Ile Val Leu Gly Pro Gln AspPhe Leu 50 55 60 Arg Thr Ala Leu Ala Ser Gly Glu Leu Val Arg Val Leu ProGlu Phe 65 70 75 80 Glu Ala Pro Ser Arg Ser Met His Leu Val Tyr Thr AlaAsn Arg Gln 85 90 95 Arg Thr Ala Lys Leu Arg Cys Phe Val Glu Thr Val LeuGly Arg Phe 100 105 110 Gly Pro Val 115 5 114 PRT Unknown MicrobialOrganism from the human gut 5 Arg Thr Arg Arg Ile Ser Leu Pro His LysLys Leu Ala Arg Asn Gly 1 5 10 15 Val Leu Tyr Ser His Gly Ala Thr GlnGlu Asp Ile Phe Ala Pro Cys 20 25 30 Gln His Arg Arg Cys Gln Ile Thr LysAla Tyr His Glu Ala Arg Leu 35 40 45 Val Glu Gln Ser Arg Arg Gln Arg ThrAla Leu Gln His Pro His Gln 50 55 60 Arg Leu Lys Leu Ser Arg Thr Pro ArgHis Met Gln Asp Val Gly Cys 65 70 75 80 Val Ala Leu Thr Gly Gly Leu GlnAla Ala Lys Asp Leu Ser His Gln 85 90 95 Ser Thr Lys Thr Arg Tyr Ser ProAla Gly Gly His Arg Asp Gly Pro 100 105 110 Xaa Val 6 190 PRTClostridium pasteurianum 6 Met Asn Lys Thr Lys Asp Asn Ile Phe Tyr SerAla Ile Lys Val Phe 1 5 10 15 Ser Asn Asn Gly Tyr Asn Gly Ala Thr MetAsp Glu Ile Ala Ser Asn 20 25 30 Ala Gly Val Ala Lys Gly Thr Leu Tyr TyrHis Phe Lys Ser Lys Glu 35 40 45 Glu Ile Phe Lys Tyr Ile Ile Glu Glu GlyVal Asn Leu Met Lys Asn 50 55 60 Glu Ile Asp Glu Ala Thr Asp Lys Glu LysThr Ala Leu Glu Lys Leu 65 70 75 80 Lys Ala Val Cys Arg Val Gln Leu AsnLeu Ile Tyr Lys Asn Arg Asp 85 90 95 Phe Phe Lys Val Ile Ala Ser Gln LeuTrp Gly Lys Glu Leu Arg Gln 100 105 110 Leu Glu Leu Arg Asp Ile Met ArgAsn Tyr Val Val His Ile Glu Glu 115 120 125 Phe Val Lys Asp Ala Met GluAla Gly Ser Ile Lys Lys Gly Asn Ser 130 135 140 Leu Phe Val Ala Tyr AlaPhe Leu Gly Thr Leu Cys Ser Val Ser Leu 145 150 155 160 Tyr Glu Val IleAsn Ala Glu Asn Asp Asn Ile Asn Asn Thr Ile Glu 165 170 175 Asn Leu MetAsn Tyr Ile Leu Asn Gly Ile Gly Leu Gln Asn 180 185 190 7 200 PRTMycobacterium tuberculosis 7 Met Asp Arg Val Ala Gly Gln Val Asn Ser ArgArg Gly Glu Leu Leu 1 5 10 15 Glu Leu Ala Ala Ala Met Phe Ala Glu ArgGly Leu Arg Ala Thr Thr 20 25 30 Val Arg Asp Ile Ala Asp Gly Ala Gly IleLeu Ser Gly Ser Leu Tyr 35 40 45 His His Phe Ala Ser Lys Glu Glu Met ValAsp Glu Leu Leu Arg Gly 50 55 60 Phe Leu Asp Trp Leu Phe Ala Arg Tyr ArgAsp Ile Val Asp Ser Thr 65 70 75 80 Ala Asn Pro Leu Glu Arg Leu Gln GlyLeu Phe Met Ala Ser Phe Glu 85 90 95 Ala Ile Glu His His His Ala Gln ValVal Ile Tyr Gln Asp Glu Ala 100 105 110 Gln Arg Leu Ala Ser Gln Pro ArgPhe Ser Tyr Ile Glu Asp Arg Asn 115 120 125 Lys Gln Gln Arg Lys Met TrpVal Asp Val Leu Asn Gln Gly Ile Glu 130 135 140 Glu Gly Tyr Phe Arg ProAsp Leu Asp Val Asp Leu Val Tyr Arg Phe 145 150 155 160 Ile Arg Asp ThrThr Trp Val Ser Val Arg Trp Tyr Arg Pro Gly Gly 165 170 175 Pro Leu ThrAla Gln Gln Val Gly Gln Gln Tyr Leu Ala Ile Val Leu 180 185 190 Gly GlyIle Thr Lys Glu Gly Val 195 200 8 192 PRT Auifex aeolicus 8 Met Tyr IleLeu Leu Phe Met Gly Glu Lys Arg Ser Asp Thr Lys Glu 1 5 10 15 Lys IleLeu Ser Ser Ala Leu Lys Leu Phe Ser Lys Lys Gly Phe Lys 20 25 30 Glu ThrThr Ile Lys Asp Ile Ala Lys Glu Val Gly Ile Thr Glu Gly 35 40 45 Ala IleTyr Arg His Phe Thr Ser Lys Glu Glu Ile Ile Lys Ser Leu 50 55 60 Leu GluSer Ile Thr Lys Glu Leu Arg His Lys Leu Glu Val Ala Leu 65 70 75 80 GlnArg Gly Glu Thr Asp Glu Glu Ile Leu Glu Ser Ile Val Asp Thr 85 90 95 LeuIle Asp Tyr Ala Phe Ser Asn Pro Glu Ser Phe Arg Phe Leu Asn 100 105 110Leu Tyr His Leu Leu Lys Glu Tyr Gly Glu Val Lys Asn Leu Pro Gly 115 120125 Glu Leu Ile Leu Lys Phe Leu Asn Gly Leu Tyr Leu Lys Arg Lys Leu 130135 140 Lys Thr Tyr Pro Glu Ile Ala Leu Ala Val Val Thr Gly Ser Val Glu145 150 155 160 Arg Val Phe Ile Phe Lys Glu Arg Asn Phe Leu Asp Tyr AspGlu Glu 165 170 175 Thr Ile Lys Lys Glu Leu Lys Lys Val Leu Lys Ser AlaIle Leu Ala 180 185 190 9 18 DNA Unknown Microbial Organism from thehuman gut 9 ccgtgggcat ccagtccg 18 10 19 DNA Unknown Microbial Organismfrom the human gut 10 tctgctcata cacgtcacg 19

We claim:
 1. An isolated inflammatory bowel disease associated I-2polypeptide, comprising substantially the same amino acid sequence asSEQ ID NO:
 2. 2. The isolated I-2 polypeptide of claim 1, comprising theamino acid sequence SEQ ID NO:
 2. 3. An isolated immunoreactive fragmentof an I-2 polypeptide, comprising substantially the same amino acidsequence as a portion of an I-2 polypeptide (SEQ ID NO: 2).
 4. Theisolated immunoreactive fragment of claim 3, comprising at least tencontiguous amino acids of SEQ ID NO:
 2. 5. Substantially purifiedantibody material that selectively binds an I-2 polypeptide having SEQID NO:
 2. 6. The substantially purified antibody material of claim 5,which is monoclonal antibody material.
 7. An isolated nucleic acidmolecule, comprising a nucleic acid sequence encoding substantially thesame amino acid sequence as SEQ ID NO:
 2. 8. The isolated nucleic acidmolecule of claim 7, comprising the nucleic acid sequence SEQ ID NO: 1.9. A method of diagnosing inflammatory bowel disease (IBD) in a subject,comprising: (a) obtaining a sample from said subject; (b) contactingsaid sample with an I-2 polypeptide, or immunoreactive fragment thereof,under conditions suitable to form a complex of said I-2 polypeptide, orsaid immunoreactive fragment thereof, and antibody to said I-2polypeptide; and (c) detecting the presence or absence of said complex,wherein the presence of said complex indicates that said subject hasIBD.
 10. The method of claim 9, wherein the presence or absence of saidcomplex is detected with a detectable secondary antibody that hasspecificity for a class determining portion of said antibody to said I-2polypeptide.
 11. The method of claim 9, wherein said IBD is Crohn'sdisease.
 12. The method of claim 9, wherein said I-2 polypeptidecomprises the sequence SEQ ID NO:
 2. 13. A method of inducing tolerancein a patient with IBD, comprising administering an effective dose of I-2polypeptide, or tolerogenic fragment thereof, to said patient with IBD.14. The method of claim 13, wherein said IBD is Crohn's disease.
 15. Themethod of claim 13, wherein said I-2 polypeptide comprises the sequenceSEQ ID NO:
 2. 16. A composition comprising an I-2 polypeptide havingsubstantially the same amino acid sequence as SEQ ID NO: 2, ortolerogenic fragment thereof, combined with a tolerogizing molecule. 17.The composition of claim 16, wherein said I-2 polypeptide has the aminoacid sequence SEQ ID NO:
 2. 18. The composition of claim 16, whichcomprises a tolerogenic fragment having at least ten contiguous aminoacids of SEQ ID NO:
 2. 19. A method of identifying an agent useful intreating inflammatory bowel disease (IBD), comprising: (a) obtaining aspecimen of an enteric bacteria from a patient with IBD; (b) isolatingfrom said specimen a microbial species that comprises a nucleic acidmolecule encoding an I-2 polypeptide; (c) contacting said microbialspecies with an agent; and (d) assaying for reduced growth or viabilityof said microbial species as compared to the growth or viability in theabsence of said agent, wherein said reduced growth or viability of saidmicrobial species indicates that said agent is an agent useful intreating IBD.
 20. The method of claim 19, wherein said IBD is Crohn'sdisease.
 21. The method of claim 19, wherein said agent is anantibiotic.
 22. A method of identifying an agent useful in treatinginflammatory bowel disease (IBD), comprising: (a) administering I-2polypeptide to a non-human animal, whereby one or symptoms of IBD areexhibited; (b) administering an agent to said non-human animal; and (c)assaying the level of said one or more symptoms characteristic of IBD,wherein a reduction in the level of said one or more symptoms ascompared to a control level indicates that said agent is an agent usefulin treating IBD.
 23. The method of claim 22, wherein said IBD is Crohn'sdisease.
 24. The method of claim 22, wherein said I-2 polypeptide hasthe amino acid sequence SEQ ID NO:
 2. 25. The method of claim 22,wherein said non-human animal is a mouse selected from the groupconsisting of a Gαi2 −/− mouse; a TCRα −/− mouse and a IL-10−/− mouse.